This invention relates to the production of phosphopeptides having anticariogenic and other properties from casein.
In our Australian Patent No. 593365, we have described that four of the many phosphopeptides released by tryptic digestion of casein have anticariogenic (tooth-decay-inhibiting) activity. These peptides all contain the active sequence -Ser(P)-Ser(P)-Ser(P)-Glu-Glu- and correspond to xcex1s1 (59-79) SEQ.ID No 2 (T1), xcex2(2-25) SEQ.ID No 3 (T2), xcex1s2(46-70) SEQ.ID No 4 (T4) and xcex1s2(2-21) SEQ.ID No 7 (T3). The methods described for the production of the anticariogenic phosphopeptides are selective precipitation and ion exchange chromatography. While these methods produce very pure preparations of these peptides, they have not received general acceptance in the dairy industry due to their cost and the level of technical skill required.
Recently membrane ultrafiltration has found broad acceptance in the dairy industry for milk treatment. In U.S. Pat. Nos. 4,358,465, 4,361,587 and 4,495,176, Brule et al describe an ultrafiltration method for the production of casein phosphopeptides as dietetic aliments. This procedure proves unsuitable for the production of anticariogenic phosphopeptides due to the predominance of non-anticariogenic phosphopeptides in the preparations.
In AU-B 66783/81 and 51491/85 Brule et al, a method of extracting phosphopeptides for use as nutritional complements is disclosed in which an aggregate forming bivalent cation is used in combination with ultrafiltration followed by diafiltration with water to extract the desired phosphopeptides selected for the above purpose. This procedure is similarly unsuitable for the production of anticariogenic phosphopeptides since diafiltration with water results in the deaggregation of the anticariogenic phosphopeptides.
It is an object of the present invention to provide a method of preparing selected phosphopeptides from casein using ultrafiltration.
The invention provides a method for the preparation of selected phosphopeptides comprising the steps of completely digesting a soluble monovalent cation salt of casein in solution, introducing a di or trivalent metal ion to cause aggregation of at least the selected phosphopeptides in said digested solution, and diafiltering the solution containing the aggregation ion through a filter having a molecular weight exclusion limit selected to retain at least said aggregated phosphopeptides while passing the bulk of the remaining phosphopeptides and non-phosphorylated peptides.
In the methods described by Brule et al, the object is to obtain a broad range of phosphopeptides from casein for use as a dietetic aliment. Therefore Brule et al do not each that the hydrolysed casein compound must be diafiltrated in the presence of the aggregating ion to ensure that the selected phosphopeptides are filtered from the solution while allowing the remaining phosphopeptides and non-phosphorylated peptides to pass during the diafiltration process. This represents a significant advance in the art since it enables the use of an industry accepted method of extraction which results in a preparation which is rich ( greater than 90% w/w) in the desired phosphopeptides.
In a preferred form of the invention, the selected phosphopeptides are the anticariogenic phosphopeptides referred to above, and the molecular exclusion limit adopted during the filtering step of the above method preferably substantially falls within the range 10,000 to 20,000.
The soluble monovalent cation salt of casein, such as sodium caseinate or potassium caseinate, may be present in the solution in a concentration substantially falling within the range 0.1 to 50% w/w, which is preferably digested using a proteolytic enzyme, such as pancreatin, trypsin, papain or chymotrypsin, or a mixture of proteolytic enzymes such as trypsin and chymotrypsin or by chemical means, such as cyanogen bromide. The enzyme(s) to casein ratio can range from about 1:1000 to 1:10 (w:w) but this would be selected to allow complete digestion of the casein as defined above. The pH of the hydrolysis should preferably be controlled at optimum for the enzymes to allow complete casein digestion. The temperature also should be optimised for complete digestion but temperature induced degradation (deamidation, dephosphorylation and peptidolysis) should be minimised. The optimal temperature is between about 20xc2x0 C. and 60xc2x0 C.
In a preferred form of the invention, after digestion HCl is added at room temperature to about pH 4.7 and any precipitate (this should be minimal) removed. CaCl2 is then added to the supernatant to a level of about 1.0% w/v. Phosphopeptides in the presence of 1.0% w/v calcium (II) aggregate. The anticariogenic phosphopeptides (ie. containing the sequence -Ser(P)-Ser(P)-Ser(P)-Glu-Glu-) form hexamers which are separated from the smaller non-anticariogenic phosphopeptide aggregates by extensive diafiltration through a 10,000 molecular weight exclusion limit filter with a CaCl2 solution preferably 1.0% w/v. The preferred molecular weight exclusion limit of the membrane filter should not be less than 10,000 or greater than about 20,000. The addition of a CaCl2 solution, or some other suitable di/trivalent metal ion, such as zinc (II) or ferric (III), is essential for diafiltration in order to maintain the integrity of the anticariogenic phosphopeptide aggregates thus allowing separation of the anticariogenic from the non-anticariogenic phosphopeptides.
After several volumes of 1.0% CaCl2 w/v have passed through the membrane filter to achieve greater than 90% purity of the anticariogenic phosphopeptides the ultraretentate containing the anticariogenic phosphopeptides can be diafiltered with water through a 1,000 molecular weight exclusion limit filter to remove calcium if desired. The retentate is then concentrated and spray dried.
The calcium, zinc and ferric salts of the anticariogenic phosphopeptide preparation (ACPP) can be converted to a sodium salt by acidifying a 10% w/v solution of the calcium ACPP to a low pH, circa pH 2.0, with HCl. After extensive diafiltration through a 1,000 molecular weight exclusion limit filter the retentate is neutralised to pH 7.0 with NaOH and then diafiltered with water through the same filter to remove excess sodium chloride.
The calcium ACPP can be converted to calcium phosphate ACPP by addition of CaCl2 and Na2HPO4 where the Ca/P final ratio is 1.67. The peptide xcex1s1(59-79) can bind 21 Ca and 13 PO4. The filtrate of the above process is suited for the purification of other bioactive casein peptides by size and charge-based separation technologies and can be used as microbiological growth media, as dietary supplements after debittering or as a nitrogen fertilizer.